A micro-perfusion chamber for single-cell fluorescence measurements

Abstract A versatile closed micro-perfusion chamber designed for single-cell fluorescence measurements under maximum microscopic magnification is described. Glass coverslips with adherent cells can be attached to the top or bottom of the chamber, depending on whether an inverted or an upright microscope is used. Eight conical holes drilled in the side of the chamber serve for the insertion of plugs with attachments for perfusion, rapid injection of small amounts of reagents, temperature measurements or for heating the interior of the chamber. Materials used in the construction of the chamber are non-toxic and resistant to standard sterilization procedures. Perfusion and temperature properties of the chamber are described. Single cell fluorescence measurements are presented in human monocyte-derived macrophages in which NAD(P)H and intracellular calcium are measured

The role of interleukin-21 in COVID-19 vaccine–induced B cell–mediated immune responses in patients with kidney disease and kidney transplant recipients

T-cell–mediated help to B cells is required for the development of humoral responses, in which the cytokine interleukin (IL)-21 is key. Here, we studied the mRNA-1273 vaccine–induced SARS-CoV-2–specific memory T-cell IL-21 response, memory B cell response, and immunoglobulin (Ig)G antibody levels in peripheral blood at 28 days after the second vaccination by ELISpot and the fluorescent bead–based multiplex immunoassay, respectively. We included 40 patients with chronic kidney disease (CKD), 34 patients on dialysis, 63 kidney transplant recipients (KTR), and 47 controls. We found that KTR, but not patients with CKD and those receiving dialysis, showed a significantly lower number of SARS-CoV-2–specific IL-21 producing T cells than controls (P < .001). KTR and patients with CKD showed lower numbers of SARS-CoV-2–specific IgG–producing memory B cells when compared with controls (P < .001 and P = .01, respectively). The T-cell IL-21 response was positively associated with the SARS-CoV-2–specific B cell response and the SARS-CoV-2 spike S1–specific IgG antibody levels (both Pearson r = 0.5; P < .001). In addition, SARS-CoV-2–specific B cell responses were shown to be IL-21 dependent. Taken together, we show that IL-21 signaling is important in eliciting robust B cell–mediated immune responses in patients with kidney disease and KTR